DNA Fingerprinting for Identification of Plant Species

DNA Fingerprinting for Identification of Plant Species ABSTRACT Conservation of plant resources prevents the loss of valuable plant species in the past centuries. Many species like that of wild Panax ginseng, Panax quinquefolius , Japonica are endangered and requires restoration. Its adverse impact on environmental and socioeconomic values has triggered the studies on plant diversity. It is seen that appropriate identification and characterization of plant materials is essential for the conservation of plant resources and to ensure their sustainable use. Molecular tools developed in the past few years provide easy, less laborious means for assigning known and unknown plant taxa. These techniques answer many new evolutionary and taxonomic questions, which were not previously possible with only phenotypic methods. Various techniques such as DNA bar coding, random amplified polymorphic DNA (RAPD), microsatellites, amplified fragment length polymorphism (AFLP) and single nucleotide polymorphisms (SNP) have recently been used for plant diversity studi es. Sequencing based molecular techniques provide better resolution at intra-genus. Whereas data from markers such as random amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP) and microsatellites provide the means to classify individual medicine. In addition DNA methods are reliable approaches towards authentication of Chinese medicinal materials. For future reference, it is necessary to compile library of Chinese medicines which include genetic information, especially for endangered species and those with high market value and or with possible poisonous adulterants which can affect quality of medicine. INTRODUCTION For the sustainable development and for improvement and maintenance of agricultural and forestry production there is a use for conservation of plant genetic resources. The objective of plant genetic resources conservation is to preserve as broad a sample of the extant genetic diversity of target species as is scientifically and economically feasible, including currently recognized genes, traits and genotypes [1]. Genetic diversity finds its natural resources in wild species for which it is important to find out the amount of genetic variability by the way of morphological, biochemical and molecular markers, besides some interesting physiological turns. Characterization of diversity is based on morphological traits. However, it is seen that morphological variability is often restricted, characters may not be obvious at all stages of the plant development. Identification plays a very important role in diversity studies. Accurate classification of individuals is essential for evaluation of species diversity. The identification of taxonomic units and endangered species, whose genetic constitution is distinct from their more abundant relatives, is important in the development of appropriate conservation strategies Nowadays, a variety of different genetic markers has been proposed to assess genetic variability. Molecular tools provide valuable data on diversity through their ability to detect variation at the DNA level CONSERVATION OF PLANT GENETIC RESOURCES: Effective conservation of plant genetic resources requires a complementary approach which makes use of both ex situ and in situ conservation methods to maximize the genetic diversity available for use. ex situ conservation: The objective of ex situ conservation is to maintain the accessions without change in their genetic constitution [1]. The methods that are designed are such that can be used to minimize the possibility of mutation, random genetic drift, selection or contamination. It is seen that storing of seeds at low temperatures and humidities can bring long term ex situ conservation. But there are many clonally propagated species, such as banana and potato, cannot be conserved in this way, and many species, particularly tropical forest tree species, produce seeds that are recalcitrant and cannot be stored. These species can only be maintained ex situ in field gene banks as growing collections of plants, or in vitro using tissue culture or cryopreservation [2]. In situ conservation: In situ conservation is considered to be the method of choice for conserving forest species and wild crop relatives and there is increasing interest in the use of in situ conservation for crops themselves (on-farm conservation) [3]. In situ conservation allows evolution to continue, increases the amount of diversity that can be conserved, and strengthens links between conservation workers and the communities who have traditionally maintained and used the resources. All genetic resources conservation activities require characterization of the diversity present in both the gene pools and the gene banks. Molecular genetics has an important role to play in many aspects of conservation such as characterizing plant genetic diversity for purposes of improved acquisition, maintenance and use. A number of different techniques are available for identifying genetic differences between organisms. The choice of technique for any one specific use will depend upon the material being studied and the nature of the questions being addressed. Protein polymorphisms were the first markers used for genetic studies. However, the number of polymorphic loci that can be assayed, and the level of polymorphisms observed at the loci are often low, which greatly limits their application in genetic diversity studies. With the development of new technologies, DNA polymorphisms have become the markers of choice for molecularâ‚ ¬Ã‚ based surveys of genetic variation. DNA ma rkers are useful in both basic (e.g. phylogenetic analysis and search for useful genes) and applied research (e.g. marker assisted selection, paternity testing and food traceability). A number of markers are now available to detect polymorphisms in nuclear DNA [4]. Properties desirable for ideal DNA markers include highly polymorphic nature, co dominant, frequent occurrence in the genome, selective neutral behavior, easy access, easy and fast assay and high reproducibility [5]. NEED FOR GENETIC RESOLUION: It is a duty of Gene bank managers and conservationists concerned with both in situ and ex situ management to conserve as much as possible the extinct genetic diversity of the species with which they work. The effectiveness with which they do this depends to a large extent on the genetic information available on the germplasm with which they work. Molecular markers provide genetic information of direct value in key areas of conservation both ex situ and in situ. For ex situ conservation the key issues are: Acquisition: Data on the diversity of existing collections can be used to plan collection and exchange strategies. In particular, calculations of genetic distances based on molecular data can be used to identify particular divergent subpopulations that might harbour valuable genetic variation that is under-represented in current holdings Maintenance: Genetic data are essential to identify duplicate accessions in order to ensure best use of available resources. Genetic markers are also needed to monitor changes in genetic structure as accessions are generated. Molecular markers provide markers suitable for both of these. Characterization: The genetic diversity within collections must be assessed in the context of the total available genetic diversity for each species. Existing passport data document the geographic location where each accession was acquired. However, passport records are often missing or incorrect. Molecular markers may extend and complement characterization based on morphological or biochemical descriptions, providing more accurate and detailed information than classical phenotypic data. Distribution to users: Users of collections benefit from genetic information that allows them to identify valuable traits and types quickly. On a more fundamental level, molecular marker information may lead to the further identification of useful genes contained in collections. Molecular data on diversity may provide essential information to develop core collections [6] that accurately represent the entire collection. Molecular markers may therefore be used in four types of measurements needed for effective ex situ conservation, all of which are useful in resolving the numerous operational, logistical, and biological questions that face gene banks managers [7]. These are: identity: the determination of whether an accession or individual is catalogued correctly, is true to type, maintained properly, and whether genetic change or erosion has occurred in an accession or population over time; Similarity: the degree of similarity among individuals in an accession or between accessions within a collection. Structure: the partitioning of variation among individuals, accessions, populations, and species. Genetic structure is influenced by in situ demographic factors such as population size, reproductive biology and migration. Detection: the presence of particular allele or nucleotide sequence in a taxon, gene bank accession, in situ population, individual, chromosome or cloned DNA segment. Those concerned with in situ conservation need to ensure that appropriate populations are identified and managed in such a way that they survive and continue to evolve. Their responsibilities can include: Location: the identification of populations which should be conserved based on the genetic diversity present as well as on the value of the resource and the threats to it. Crucial to this is knowledge of the extent and distribution of genetic diversity in species populations which should optimally include molecular data. Management: the development of management plans to monitor the changes in target populations over time and ensure their continued survival. The populations maintained in situ constitute part of ecosystems and both intra- and interspecific diversity must be maintained over time at appropriate levels. Accessibility: in situ conservation is most commonly of interest in forest genetic resources conservation and that of wild crop relatives but it is also of increasing interest for on-farm conservation of traditional cultivars. Genetic resources conserved in this way remain accessible to the communities who depend on them. Managers need to ensure they are also accessible to other users and that sufficient genetic information is available to assist such users. Within the context of in situ conservation, therefore, identity, similarity, structure and detection are also important and can be usefully investigated using molecular techniques BASIC GENETIC TOOLS DNA sequencing: DNA sequencing is the determination of the precise sequence of nucleotides in a sample of DNA. The nucleotides bases are A (adenine), G (guanine), C (cytosine) and T (thymine) The conventional and next generation sequencing techniques are thus been explained in detail. Conventional Sequencing Technique- Now days it is seen that dye-terminator sequencing technique is the standard method in automated sequencing analysis [8]. And for majority of sequencing the dye-terminator sequencing method, along with automated high-throughput DNA sequence analyzers, is used. Dye-terminator sequencing  utilizes labelling of the chain terminator dents, which permits sequencing in a single reaction, rather than four reactions as in the labelled-primer method. In dye-terminator sequencing, each of the four di de-oxynucleotide chain terminators is labelled with fluorescent dyes, each of which emit light at different wavelengths. Owing to its greater expediency and speed, dye-terminator sequencing is now the mainstay in automated sequencing. The main advantages of this technique are its robustness, automation and high accuracy Its limitations include dye effects due to differences in the incorporation of the dye-labelled chain terminators into the DNA fragment, resulting in unequal peak heights and shapes in the electronic DNA sequence trace  chromatogram  after  capillary electrophoresis  . This problem has been addressed with the use of modified DNA polymerase enzyme systems and dyes that minimize incorporation variability, as well as methods for e liminating dye blobs. DNA barcoding of plants has now gained the interest of scientists with the aim to identify an unknown plant in terms of a known classification. DNA barcoding is a technique for characterizing species of organisms using a short DNA sequence from a standard. DNA barcode sequences are thus shorter than the entire genome and can be obtained quickly [9]. Basic Local Alignment Search Tool (BLAST) was used for species-level assignment of plants and individual barcodes were obtained with matK (99%), followed by trnH-psbA (95%) and then rbcL (75%) [10]. Recently, a group of plant DNA barcode researchers proposed two chloroplast genes, rbcL and matK, taken together, as appropriate for bar-coding of plants [11]. Chloroplast DNA (cpDNA) is the basis of Molecular phylogenies in plants but the problems due to gene flow of cpDNA among closely related taxa, as well as the lack of phylogenetic resolution, triggered the development of new approaches based on nuclear DNA [13]. The most common alternative corresponds to the sequencing of the ITS (internal transcribed spacer) of 18S-25S nuclear ribosomal DNA [14, 15]. The failure of both cpDNA and ITS techniques to sequence, the amplified fragment length polymorphism (AFLP) approach has the potential to solve such difficulties, particularly among closely related species, or at the intra-specific level [16-18]. Therefore, integration of recently developed bar-coding with the following techniques such as RAPD, AFLP, microsatellite and SNP seems to provide better resolution. Next Generation Sequencing Techniques Next generation platforms do not rely on Sanger chemistry [19] as did the first generation machines used for the last 30 years. The first of this kind of 2nd generation of sequencing technique appeared in 2005 that was based on pyrosequencing [20, 21] Commercial 2nd generation sequencing methods can be distinguished by the role of PCR in library preparation. There are four main platforms; all being amplification-based: (i) Roche 454 GS FLX, (ii) Illumina Genome Analyzer IIx, (iii) ABI SOLiD 3 Plus System and (iv) Polonator G.007 [22] The single-molecule sequencing method (also known as 3rd generation or next-next generation) is independent of PCR [25,30]. This mode of sequencing protocol was recently developed by Helicos Genetic Analysis System using the technology developed by Braslavsky et al. [23]. Other 3rd generation sequencing systems are being developed by Life Technologies and Pacific Biosciences SMRT technology and may appear within one to two years. Random Amplified Polymorphic DNA (RAPD) The invention of PCR (polymerase chain reaction) is a milestone in the development of molecular techniques. PCR results in the selective amplification of a chosen region of a DNA molecule. Random amplification of DNA with short primer by PCR is a useful technique in phylogenetics. The important point is the banding pattern seen, when the products of PCR with random primers are electrophoresed in a reflection of the overall structure of the DNA molecule used as the template. If the starting material is total cell DNA then the banding pattern represents the organization of the cells genome. Differences between the genomes of two organisms can be measured with RAPD. Two closely related organisms would be expected to yield more similar banding patterns than two organisms that are distant in evolutionary terms [24]. Moreover, this technique requires only small piece of animal tissue or blood, as the extracted DNA can be amplified million times using PCR. Basic protocol: EXTRACTION OF HIGHLY PURE DNA ADDITION OF SINGLE ARBITARY PRIMER POLYMERASE CHAIN REACTION (PCR) SEPARATION OF FRAGMENTS BY GEL ELECTROPHORESIS VISUALIZATION OF RAPD-PCR FRAGMENTS AFTER EtBr STAINING UNDER UV DETERMINATION OF FRAGMENT SIZE This technique has mainly gained attraction as there is no requirement for DNA probes or sequence information for primer designing. There are also no blotting or hybridizing steps. This technique only requires the purchase of a thermo cycling machine and agarose gel apparatus and relevant chemicals, which are available as commercial kits and also it is a quick and simple technique. It is important to note that RAPD technique requires maintaining strictly consistent reaction conditions in order to achieve reproducible profiles [25]. The RAPD markers have been used for detecting genomic variations within and between varieties of sweet potato. A total of 160 primers were tested and eight showed consistent amplified band patterns among the plants with variations within and between varieties [26] of sweet potato. Restriction fragment length polymorphism All organisms are genotypically different because they have had numerous differences in their genomic DNA. This difference results in a restriction fragment length polymorphism. Here the chromosomal DNA is first cleaved by restriction enzymes creating fragments and then these fragments are separated by agarose gel electrophoresis. After it southern hybridization analysis is carried out using probe that spans the region of interest. The probe hybridizes to the relevant region, lighting up the appropriate restriction fragments on the resulting autoradiograph. If an RFLP is present then it will be clearly visible on the autoradiograph. Thus RFLP is used as a major tool to identify the genetic diversity within and between species [27]. Basic Protocol CHROMOSOMAL DNA Cleave with Restriction enzymes DNA FRAGMENTS SEPARATE FRAGMENTS BY AGAROSE GEL ELECTROPHORESIS DENATURE DNA AND TRANSFER TO NITROCELLULOSE Radiolabelled DNAprobe INCUBATE WITH PROBE EXPOSE X-RAY FILM TO PAPER Amplified fragment length polymorphism AFLP analysis is able to detect high levels of polymorphism and has high repeatability and speed of analysis. AFLP technique as being based on the detection of restriction fragments by PCR amplification and argued that Ê ¹the reliability of the RFLP technique is combined with the power of the PCR techniqueÊ ¹. Firstly extraction of highly purified DNA then restriction endonuclease digestion of DNA followed by ligation of adapters. After this amplification of these fragments is done by two primers, and then gel electrophoresis and analysis of fragments by automated sequencing machines. The advantage of this technique is that it is applicable to all species and unlike RAPD; this technique is highly reproducible as it combines restriction digestion and PCR. However, AFLP requires more DNA (300-1000 ng per reaction) and is more technically demanding than RAPD [4].AFLP markers in surveys of plant diversity are discussed in a review published by Mba and Tohme [28]. Recently, Jatropha curcas [29] and Rhodiola rosea [30] have been characterized by AFLP in germplasm collection. The wild populations of Agave angustifolia in the desert was studied by Teyer et al. [31] using AFLP to measure the genetic variability within and between natural populations. AFLP markers have been extensively used for phylogenetic analysis and determining the genetic diversity for conservation of endangered plant species [32-36]. ISOLATION OF GENOMIC DNA Basic protocol: DIGESTION WITH ONE OR MORE RESTRICTION ENZYMES LIGATION OF RESTRICTION HALF-SITE SPECIFIC ADAPTORS TO ALL RESTRICTION FRAGMENTS AMPLIFICATION OF THESE FRAGMENTS WITH TWO PCR PRIMERS THAT HAVE CORRESPONDING ADAPTORS AND RESTRICTION SPECIFIC SEQUENCES ELECTROPHORETIC SEPARATION OF AMPLICONS ON A GEL MATRIX MICROSATELLITES Microsatellites, are alternatively known as simple sequence repeats (SSRs), short tandem repeats (STRs) or simple sequence length polymorphisms (SSLPs). These are tandem repeats of sequence units generally less than 5 bp in length [37].One common example of a microsatellite is a (CA)n repeat, where n is variable between alleles. These markers often present high levels of interâ‚ ¬Ã‚  and intraâ‚ ¬Ã‚ specific polymorphism, particularly when tandem repeats number ten or greater. CA nucleotide repeats are very frequent in human and other genomes, and present every few thousand base pairs. Interâ‚ ¬Ã‚ SSRs are a variant of the RAPD technique, although the higher annealing temperatures probably mean that they are more rigorous than RAPDs. The microsatellite protocol is simple, once primers for SSRs have been designed. The first stage is a PCR, depending upon the method of detection one of the primers is fluorescently or radioactively labeled. The PCR products are separated on high resolution polyacrylamide gels, and the products detected with a fluorescence detector (e.g. automated sequencer) or an Xâ‚ ¬Ã‚ ray film. The investigator can determine the size of the PCR product and thus how many times the short nucleotide was repeated for each allele. Microsatellites developed for particular species can often be applied to closely related species, but the percentage of loci that successfully amplify may decrease with increasing genetic distance [38]. Microsatellite technique has recently been used to establish conservation strategy of endangered plants like Calystegia soldanella [39], Tricyrtis ishiiana [40] and Galium catalinense subspecies acrispum [41]. ISOLATION OF GENOMIC DNA Basic protocol: SEQUENCING DESIGNING OF PRIMERS FOR REGIONS FLANKING MICROSATELLITES ELECTROPHORETIC SEPRATION OF AMPLICONS ONA GEL MATRIX ISOLATION OF GENOMIC DNA Conclusion Molecular characterization can play a role in uncovering the history, and estimating the diversity, distinctiveness and population structure. Awareness of the level of genetic diversity and the proper management of genetic resources are important issues in modern scenario. New markers deriving from DNA technologies are valuable tools to study genetic variability for conservation purposes. In the near future, the advent of genomics will give an impressive tool for genetic resources evaluation.